PROJECT SUMMARY Sleep and circadian disruption are highly prevalent in Alzheimer?s disease, emerging decades prior to cognitive decline. Evidence from animals and humans suggests these disruptions directly lead to Alzheimer?s disease pathology that further exacerbate sleep and circadian dysfunctions. An important breakthrough for studying these connections was the recent development of a genetically diverse mouse panel that incorporates high-risk familial Alzheimer?s disease mutations (termed AD-BXD) that recapitulate key aspects of human Alzheimer?s pathophysiology, including aging-related neurodegeneration, progressive cognitive deficits, and sleep disruption. Using these mice, identified a new marker of genetic vulnerability to cognitive decline and sleep disruption in Alzheimer?s disease: the transient-receptor potential nonselective cation channel type 3 (TRPC3). TRPC3 has already been implicated as a target for modifying the development of normal cognitive aging and Alzheimer?s disease. We found that viral-mediated knockdown of TRPC3 diminished amyloid load and enhanced cognition in susceptible AD-BXD mice, providing the preclinical basis for investigating the mechanistic links that connect sleep disruption and cognitive decline in Alzheimer?s disease. However, these methods do not offer the biology by which TRPC3 moderate disease-related pathology. We propose to comprehensively map the spatial location and expression of TRPC3 to identify whether this localization changes in key brain regions related to sleep and cognition due to Alzheimer?s pathology. We developed a new approach to rapidly image multiple cell-types and markers of Alzheimer?s neurodegeneration within a whole brain in three-dimensional (3D) space at single-cell resolution. Our ultra-fast high-resolution confocal ribbon-scanning approach reaches diffraction limited resolution (~200nm) and collects 3D rendered whole brain maps in less than 24-hours. The goals of the supplement are to take advantage of the discovery of TRPC3 as a new target for Alzheimer?s-related changes in cognition and sleep and use our innovative tools to answer fundamental questions: In mice, 1) Is TRPC3 located in disease-related brain areas linked to cognition and sleep?; and 2) Does TRPC3 interact with biological hallmarks of Alzheimer?s disease brain pathology, including amyloid beta and hyperphosphorylated Tau? We will use human postmortem brains from Alzheimer?s disease patients to ask, 3) Is brain TRPC3 expression altered in Alzheimer?s disease? We predict TRPC3 localizes to subregions of the hypothalamus associated with sleep and hippocampal and cortical regions associated with cognition, and this distribution will be differentially impacted by sleep deprivation in susceptible vs. resilient mice. In human brains, we expect that TRPC3 expression will be higher in advanced Alzheimer?s disease patient brains and display altered rhythmicity. Ultimately, using our novel suite of genetic, imaging, and computational tools, we will we will answer longstanding questions about how changes in sleep and cognitive decline are linked to Alzheimer?s disease pathology.